VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

the lysyl-tRNA synthetase YjeA, the small RNA binding protein Hfq andYjeJ, a protein with an unknown function, were identified to play a role inthe regulation of the Cad system. Mutants of these genes were either unableto express the genes of the cadBA operon or - in the case of YjeJ - theexpression was higher as in the wild-type. Posttranslational modification isone interesting aspect which could be involved in the regulation process.Another aspect is the control of the acid response system at thetranscriptional level by small RNAs with the help of Hfq. It is discussedhow these components extend the regulation network of the Cad system.[1] Shi, X.et al (1993): Modulation of acid induced amino-acid decarboxylase gene expression by H-NS in Escherichia coli. J Bacteriol 175: 1182-1186.[2] Tetsch, L. et al (2008): The membrane-integrated transcriptional activator CadC of Escherichiacoli senses lysine indirectly via the interaction with the lysine permease LysP. Mol Microbiol 67: 570-583.[3] Watson, N. et al (1992): Identification of elements involved in transcriptional regulation of theEscherichia coli cad operon by external pH. J Bacteriol 174: 530-540.SRP029Osmotic stress induces different stress responses inEnterococcus faecalis and Enterococcus faeciumS. Kirchen*, M. Brändle, L. Baumgärtner, U. Obst, T. SchwartzInstitute of Functional Interfaces (IFG), Karlsruhe Institute of Technology(KIT), Karlsruhe, GermanyBacterial encounter changing environments, where they have to cope withlimited nutrients, temperature shifts and other stresses. Thus, bacterialsurvival and fitness is dependent on an adequate stress response. The stressresponses of the opportunistic pathogens Enterococcus faecium andEnterococcus faecalis in respect to osmotic stress were investigated via (i)genomic fingerprinting and (ii) gene expression analyses of a specific stressmarker. To investigate general osmotic stress induced genome alterations viagenomic fingerprinting, RAPD (randomly amplified polymorphic DNA) -PCR was applied. Short, unspecific binding primers were used in a PCRreaction and the generated fingerprints were compared. Polyphosphatekinase (PPK) encoded by ppk gene, catalyses the synthesis of polyP inbacteria and plays an important role in stress tolerance, virulence andsurvival. Expression of the ppk gene was assayed as specific stress marker.Enterococci in the early stationary growth phase were transferred into 0.5MNaCl solution and incubated for three, four and five days respectively.Osmotic stress did not change the genomic fingerprint of Enterococcusfaecium, indicating its robustness, whereas RAPD-PCR of Enterococcusfaecalis showed variations on the genome level, indicating the strainsosmosensitivity. In parallel RNA from both enterococci was extracted andtranscribed into cDNA using random hexamers. Expression analyses of theppk gene in comparison to16S ribosomal housekeeping gene wereperformed. In both enterococci the stress responsive target ppk wasconstantly expressed during salt stress application.Whereas in Enterococcusfaecalis the 16S rRNA was also constantly expressed, the amount ofribosomal 16S rRNA in Enterococcus faecium decreased significantly uponsalt stress. It is likely that the reduction of 16S is caused by ribosomaldisassembly, associated with a degradation of the ribosomal RNA. Despitetheir close relationship to each other the two enterococci show differentstress responses upon osmostress.SRP030The regulatory interplay between the membraneintegratedtranscriptional activator CadC and the lysinetransporter LysP in Escherichia coliM. Rauschmeier*, L. Tetsch, V. Schüppel, K. JungDepartment Biology I - Microbiology, Ludwig-Maximilians-UniversityMunich, Martinsried, GermanyThe Cad system is involved in the acid tolerance response of E. coli andhelps to maintain the cytosolic pH within the physiological range. Thesystem is composed of the lysine decarboxylase CadA, the lysine/cadaverineantiporter CadB and the membrane-integrated transcriptional activatorCadC. Both, the consumption of a cytoplasmic proton duringdecarboxylation of lysine and the excretion of the more alkaline polyaminecadaverine, result in an increase of the intra- and extracellular pH. CadCregulates expression of the cadBA operon and induces the transcriptionunder conditions of low external pH (5.8) and concomitantly availablelysine. CadC co-senses the exogenous lysine signal in an interplay with thelysine-specific transporter LysP. LysP inhibits CadC activation at a lowexternal lysine concentration presumably via a direct interaction with thetransmembrane domain of CadC. To gain more insights into the molecularmechanism of the interconnectivity between CadC and LysP we applied sitedirectedand random mutagenesis. Both methods generated several LysPderivatives with single amino acid replacements that altered CadC mediatedcadBA expression. To elucidate whether transport of LysP is theprerequesite for co-sensing, we investigated transport activity of thesevariants in vivo by measuring L- 14 C-lysine uptake in an E. coli strain lackingall lysine transporters. These analyses revealed a functional coupling of theregulatory and transport activities of LysP. It is still unclear, whether aminoacid replacements in LysP affect lysine binding and/or the mediation ofprotein-protein-interactions. In another approach transmembrane interactionsbetween LysP and CadC were analyzed with the BACTH system. Firstresults indicate an interaction between transmembrane helix three of LysPand the transmembrane helix of CadC.SRP031Strand specific transcriptomes of Escherichia coliO157:H7 EDL933 revealed by RNA-sequencingR. Landstorfer* 1 , S. Simon 2 , D. Oelke 2 , K. Neuhaus 1 , S. Scherer 11 Department of Microbial Ecology, Center of Life and Food SciencesWeihenstephan, Technical University Munich, Freising, Germany2 Department of Computer Sciences, Data Analysis and Visualization,University of Konstanz, Konstanz, GermanyEscherichia coli O157:H7 EDL933 is an important human pathogen.Infection leads to hemorrhagic diarrhea and can cause a hemolytic uremicsyndrome (HUS). This bacterium is transmitted by food, including produce.Its genome was sequenced in 2001. Due to the progresses in NextGeneration Sequencing we were able to sequence the total transcriptome ofthis pathogen under six different conditions. Cells were harvested from LBmedium, LB medium at pH9, LB medium with nitrite, minimal medium,homogenized spinach and the surface of raddish shoots. The sequencedtranscripts (SOLiD4.0) were mapped to the reference genome and comparedamong the six different conditions.The data give insights into gene usage under different conditions. Besidemany known genes, we have evidence for transcription of severalhypothetical genes. Owing to different expression patterns these putativegenes can now be attributed with functional involvements. In addition, thesedata sets uncovered yet unknown transcripts. Some of those show verysimilar structures to known sRNAs and asRNAs, others may code forproteins. Several genetic elements of the E. coli O157:H7 EDL933 genomecan now be re-annotated or mapped with higher precision, respectively. Thisincludes major and minor transcriptional start sites or operon configurationsunder different conditions. Taken together, the data allow to betterunderstand the mechanisms of environmental persistence and infection ofdifferent vectors.SRP032Identification of the molecular mode of action ofCarolacton, a novel biofilm inhibitorM. Reck*, B. Kunze, I. Wagner-DöblerResearch Group Microbial Communication, Helmholtz Center for InfectionResearch, Braunschweig, GermanyBiofilm forming bacteria are often significantly more resistant to drugtreatments than their planktonic counterparts and are associated to variouspathological conditions in humans as e.g. cystic fibrosis, colonisation ofindwelling medical devices and dental plaque formation. To this end newsubstances and therapies aiming to erase biofilms are urgently needed.Carolacton, a secondary metabolite isolated from the myxobacteriumSorangium cellulosum was proven to effectively kill S. mutans biofilm cellsin a wide range of concentrations while showing only minor toxic effects onplanktonically living cells [1]. A severe membrane damage, caused bycarolacton, was verified by the analysis of the protein and DNA content inthe supernatant of carolacton treated cells and the comparison to untreatedreference biofilms. Utilisation of a ß-galactosidase reporter strain revealedcytoplasmically localised ß-galactosidase to be present in large extentextracelluarly. Furthermore it was shown that carolacton interferes with theacid resistance of S. mutans. In order to evaluate the carolacton affectedtranscriptome and to get insights into the molecular mode of action acomparative time series microarray analysis using treated and untreatedbiofilm cells was performed. Up to 28% of all 1961 ORFs of S. mutans wereidentified to be differentially expressed (log FC > +/- 0.8; p < 0.001) uponcarolacton pertubation. Regulated genes include numerous coding forspektrum | Tagungsband 2011